Rabbit red blood cell hexokinase

Summary Rabbit hexokinase (EC 2.7.1.1) has been shown to exist in reticulocytes as two distinct molecular forms, designated hexokinase Ia and Ib, but only one of these was consistently present in mature red cells. In vivo, hexokinase la and Ib show a decay rate of 3 and 8% a day, respectively, while in vitro they show a similar stability.The possibility that the proteolytic activities of the reticulocyte could be responsible for the fast decay of hexokinase was investigated. No differences were found in the decay rates of hexokinase la and Ib during in vitro reticulocyte maturation in presence or absence of proteolytic inhibitors. Contrariwise, many findings indicate the ATP-dependent proteolytic system of the reticulocyte as a possible mechanism. In fact, the decay of hexokinase and the degradation of 3H-globins are both stimulated by ATP and ubiquitin; they show similar kinetic properties and both disappear during reticulocyte maturation.The cellular localization of hexokinase la and Ib was shown to be responsible for the differences found between their decay rates.Abbreviations PMSF phenylmethylsulfonyl fluoride - TPCK 1-1-tosylamide-2-phenylethyl-chloromethyl ketone - TLCK N -p-tosyl-L-lysine chloromethyl ketone     

Regulatory properties of rabbit red blood cell hexokinase at conditions close to physiological

The true level of hexokinase in rabbit erythrocytes was determined by three different methods, including the spectrophotometric glucose-6-phosphate dehydrogenase coupled assay and a new radioisotopic assay. The value found at 37°C (pH 7.2) was 10.23±1.90 μmol/h per ml red blood cells, which is lower than previously reported values. More than 40 cellular components of the rabbit erythrocytes were tested for their effects on the enzyme. Their intracellular concentrations were also determined. Several of these compounds were found to be competitive inhibitors of the enzyme with respect to Mg·ATP^2?. Furthermore, reduced glutathione at a concentration of 1 mM was able to maintain hexokinase in the reduced state with full catalytic activity. The ability of orthophosphate to remove the inhibition of some phosphorylated compounds was examined under conditions similar to cellular (pH 7.2 and 50 μM of orthophosphate) and found to be of no practical interest. In contrast, the binding of ATP^4? and 2,3-diphosphoglycerate to the rabbit hemoglobin significantly modifies their intracellular concentrations and the formation of the respective Mg complexes. The pH-dependence of the reaction velocity and of the kinetic properties of the enzyme in different buffer systems were also considered. This information was computerized, and the rate of glucose phosphorylation in the presence of the mentioned compounds was determined. The value obtained, 1.94±0.02 μmol/h per ml red blood cells, is practically identical to the measured rate of glucose utilization by intact rabbit erythrocytes (1.92±0.3 μmol/h per ml red blood cells). These results provide further evidence for the central role of hexokinase in the regulation of red blood cell glycolysis.     

Rat red blood cell hexokinase purification,properties and age-dependence

Summary Rat erythrocytes, in contrast to red blood cells from other mammals, have been shown to contain only one hexokinase isozymic form identified as type I by chromatographic and kinetic properties. Rat reticulocytes contain 3.6-times the hexokinase activity found in mature erythrocytes but exactly the same isozyme. By a combination of ion-exchange chromatography, dye-ligand chromatography and high-pressure liquid chromatography the rat erythrocyte hexokinase was purified more than 84 000-fold to a specific activity of 143 units/mg protein and shown to be homogeneous by sodium dodecyl sulfate-gel electrophoresis. The native protein showed a molecular weight of 100 000 by gel-filtration and an apparent molecular weight of 98 000 under denaturating conditions in sodium dodecyl sulfate-gel electrophoresis. The isoelectric point was shown to be 6.3 pH units. This data provides evidence of only one form of hexokinase in the erythrocytes of a mammal.     

Pig red blood cell hexokinase: Regulatory characteristics and possible physiological role

The regulatory properties of pig erythrocyte hexokinase III have been studied. Among mammalian erythrocyte hexokinases, the pig enzyme shows the highest affinity for glucose and a positive cooperative effect with n_H = 1.5 at all the MgATP concentrations studied (for 0.5 to 5 mm). Glucose at high concentrations is also an inhibitor of hexokinase III. Similarly, the apparent affinity constant for MgATP is independent of glucose concentration. Uncomplexed ATP and Mg are both competitive inhibitors with respect to MgATP. Glucose 6-phosphate, known as a stronger inhibitor of all mammalian erythrocyte hexokinases, is a poor inhibitor for the pig enzyme (K_i = 120 μm). Furthermore, this inhibition is not relieved by orthophosphate as with other mammalian red blood cell hexokinases. A variety of red blood cell-phosphorylated compounds were tested and found to be inhibitors of pig hexokinase III. Of these, glucose 1,6-diphosphate and 2,3-diphosphoglycerate displayed inhibition constants in the range of their intracellular concentrations. In an attempt to investigate the role of hexokinase type III in pig erythrocytes some metabolic properties of this cell have been studied. The adult pig erythrocyte is able to utilize 0.27 μmol of glucose/h/ml red blood cells (RBC) compared with values of 0.56–2.85 μmol/h/ml RBC for the other mammalian species. This reduced capacity to metabolize glucose results from a relatively poor ability of the cell membrane to transport glucose. In fact, all the glycolytic enzymes were present and a low intracellular glucose concentration was measured (0.5 mm against a plasma level of 5 mm). Furthermore, transport and utilization were concentration-dependent processes. Inosine, proposed as the major energy substrate of the pig erythrocyte, at physiological concentrations is not as efficient as glucose in maintaining reduced glutathione levels under oxidative stress. Furthermore, newborn pig erythrocytes (fully permeable to glucose) possess hexokinase type II as the predominant glucose-phosphorylating activity. This fact and the information derived from the study of the regulatory characteristics of hexokinase III and from metabolic studies on intact pig erythrocytes permit the hypothesis that the presence of this peculiar hexokinase isozyme (type III) enables the adult pig erythrocyte to metabolize low but appreciable amounts of glucose.     

Rabbit red blood cell hexokinase. Decay mechanism during reticulocyte maturation

In rabbit reticulocytes, the hexokinase (EC 2.7.1.1)-specific activity is 4-5 times that of corresponding mature red cells. Immunoprecipitation of hexokinase by a polyclonal antibody made in vitro shows that this maturation-dependent hexokinase decay is not due to accumulation of inactive enzyme molecules but to degradation of hexokinase. A cell-free system derived from rabbit reticulocytes, but not mature erythrocytes, was found to catalyze the decay of hexokinae activity and the degradation of 125I-labeled enzyme. This degradation is ATP-dependent and requires both ubiquitin and a proteolytic fraction retained by DEAE-cellulose. Maximum ATP-dependent degradation was obtained at pH 7.5 in the presence of MgATP. MgGTP could replace MgATP with a relative stimulation of 0.90. 125I-Hexokinase incubated with reticulocyte extract in the presence of ATP forms high molecular weight aggregates that reach a steady-state concentration in 1 h, whereas the degradation of the enzyme is linear up to 8 h, suggesting that the formation of protein aggregates precedes enzyme catabolism. These aggregates are stable upon boiling in 2% sodium dodecyl sulfate, 3% mercaptoethanol and probably represent an intermediate step in the enzyme degradation with hexokinase and other proteins covalently conjugate to ubiquitin. That hexokinase could be conjugated to ubiquitin was shown by the formation of 125I-ubiquitin-hexokinase complexes in the presence of ATP and the enzymes of the ubiquitin-protein ligase system. Thus, the decay of hexokinase during reticulocyte maturation is ATP- and ubiquitin-dependent and suggests a new physiological role for the energy-dependent degradation system of reticulocytes.     

Rabbit red blood cell hexokinase:intracellular distribution during reticulocytes maturation

Summary The intracellular localization and isozyme distribution of hexokinase were studied during rabbit reticulocyte maturation and aging. In reticulocytes 50% of the enzyme was particulate while in the mature erythrocytes all the hexokinase activity was soluble. The bound enzyme co-sediments with mitochondria and by column chromatography it was found to be hexokinase Ia. The cytosol of reticulocytes contains hexokinase Ia (38%) and hexokinase Ib (62%) while the mature erythrocytes contain only hexokinase Ia. The amount of bound hexokinase decreases very quickly during cell maturation and aging as was shown by following in vivo reticulocyte maturation or by analysis of hexokinase compartmentation in cells of different ages, obtained by density gradient ultracentrifugations. A role for this intracellular distribution of hexokinase is suggested.     

pH effects on red cell deformability

The effect of pH on erythrocyte deformability was determined in the ektacytometer. In isotonic solutions, cell deformability was essentially unaltered at pH values from 6.44 to 7.7, despite the known cell volume changes that occur over this pH range. Analysis by osmotic scan ektacytometry showed that erythrocyte deformability is homeostatically maintained constant during acidosis and alkalosis because of a balance between the countervailing effects of changes in intracellular viscosity and in the surface to volume ratio. This balance is perturbed in the sickle erythrocyte, so that acidosis improves deformability.     

Effects of rHuEPO treatment on red blood cell osmotic resistance

Red blood cell osmotic resistance (RBCOR) is defined as resistance to osmotic changes in cell integrity after their exposure to hypotonic saline solution. The investigation examined the effect of rHuEPO on RBCOR in hemodialysed patients. The study included 58 patients aged 49 +/- 14 years, treated by hemodialysis for 59 +/- 43 months on average. Half of the patients received rHuEPO for anemia correction. RBCOR was determined in all patients as 3 values: hemolysis start point (HSP), hemolysis end point (HEP) and middle osmotic resistance (MOR). The patients underwent laboratory checkup for parameters characteristically changed in the uremic syndrome. In the control group of healthy subjects (n = 16) RBCOR was only determined. No differences were found in the average values of HSP, HEP and MOR between the rHuEPO treated group of patinets and the untreated group. Compared to healthy individuals, the hemodialysed patients displayed significantly higher values of HSP, HEP and MOR. The only one significant correlation of RBCOR and routine laboratory features was found between MOR and predialytic serum concentrations of calcium (r = 0.28, p < 0.05) and hydrogen ions (r = 0.37, p < 0.05). Our results suggest that the administration of rHuEPO does not affect RBCOR in hemodialysed patients, that RBCOR is not always reduced in this population and that it correlates with a small number of laboratory parameters characteristic for the uremic syndrome.     

Effects of dextran molecular weight on red blood cell aggregation

The reversible aggregation of human red blood cells (RBC) by proteins or polymers continues to be of biologic and biophysical interest, yet the mechanistic details governing the process are still being explored. Although a depletion model with osmotic attractive forces due to polymer depletion near the RBC surface has been proposed for aggregation by the neutral polyglucose dextran, its applicability at high molecular mass has not been established. In this study, RBC aggregation was measured over a wide range of dextran molecular mass (70 kDa to 28 MDa) at concentrations ≤2 g/dL. Our results indicate that aggregation does not monotonically increase with polymer size; instead, it demonstrates an optimum dextran molecular mass around 200-500 kDa. We used a model for depletion-mediated RBC aggregation to calculate the expected depletion energies. This model was found to be consistent with the experimental results and thus provides new insight into polymer-RBC interactions.     

Effects of cell lysis on the rheological behavior of red blood cell suspensions

Rheological studies of lysed cell suspensions are performed with a magneto acoustic ball microrheometer. Two methods for lysing the cells are developed in order to provide cell volume concentrations identical to control intact cell suspensions. The first uses a freeze-thaw technique and the second uses sonication. It is found that cell suspensions disrupted by sonication have a lower viscosity than intact suspensions, whereas cell suspensions lysed by the freeze-thaw method exhibit a higher viscosity. Sonication is discovered to have a detrimental impact on the cell membrane, and to cause complete destruction of the cell membrane structure. Measurements of the steady state viscosity show that indeed the presence of the membrane is not detected, and that what is measured is mainly the viscosity of the hemoglobin solution. On the other hand, freeze-thaw results indicate that at least two phenomena occur. The first phenomenon, occurring during the first freeze-thaw cycle, produces an increase in viscosity and in viscoelasticity. The second one, taking place after subsequent freeze-thaw cycles, induces a decrease in the bulk rheological properties. Several possible mechanisms are presented to explain the observed phenomena.     

Red blood cell phagocytosis following hexokinase inactivation

Hexokinase inactivating antibodies were loaded into human erythrocytes using an encapsulation procedure based on hypotonic haemolysis, isotonic resealing and reannealing. Red blood cells loaded with anti-hexokinase IgG showed 20 percent residual hexokinase activity and reduced glycolytic activity. ⁹Incubation of these cells in the presence of an oxidizing agent such as terbutyl hydroperoxide (TBH) and then in autologous plasma, promoted opsonization by autologous IgG and complement deposition, but not haemolysis. Furthermore, the antihexokinase IgG loaded cells treated with TBH were actively recognized and phagocytosed by macrophages. Thus, metabolic impairment of human erthrocytes promotes autologous IgG binding, C3 deposition and phagocytosis, a mechanism already reported for the removal of senescent erythrocytes.     

Effects of pressure on red blood cell geometry during micropipette aspiration

Micropipette aspiration is a potentially useful and accurate technique to measure red blood cell (RBC) geometry. Individual RBCs are partially aspirated and from the resulting sphere diameter, total cell length, and pipette diameter, membrane area and cell volume can be calculated. In this study we have focused on possible shape artifacts associated with the aspirated portion of RBC. We observed that the apparent RBC geometry (calculated area and volume) changed markedly (P < 0.001) with the applied aspiration pressure; for normal human RBC the area increased by 5.6 +/- 0.6% and volume decreased by 4.7 +/- 0.6% when the aspiration pressure was increased from 20 to 100 mm water. The calculated membrane area dilation modulus was 7.4 dyn/ cm, which is far below the expected value, and microscopic observations revealed a membrane folding artifact as a possible artifact. These assumptions were strengthened by using a short-duration (3 s) pressure peak of 20-100-20 mm water. The folding then disappeared permanently, but a small (0.31 +/- 0.09%; P < 0.001) area decrease was detected which yields a realistic dilation modulus of 215 dyn/cm. We conclude that membrane folding can critically affect RBC micropipette measurements and that a transient pressure peak can unfold the RBC membrane, thus allowing accurate measurements of RBC geometry.     

Maturation dependent decline of adenylate cyclase in rabbit red blood cell membranes

Adenylate cyclase (EC 4.6.1.1) was studied in membrane preparations of reticulocyte-rich blood obtained from phenylhydrazine-treated rabbits and compared to that of untreated animals.Basal and fluoride-stimulated activities were decreased 2- and 4-fold, respectively, during the process of maturation.Catalytic parameters such as time course, protein, ATP, Mg²⁺ concentration curves and K_m have been determined and were found to be similar in the reticulocyte and the erythrocyte.Adenylate cyclase was sensitive to GTP, 5′-guanylyl imidodiphosphate, prostaglandin E₁ and prostaglandin E₂. Activation by prostaglandin E₁ was higher than that produced by prostaglandin E₂. Only additive effect was found when 5′-guanylyl imidodiphosphate or GTP was added to hormone-stimulated activity. The sensitivity of the enzyme to these effectors was decreased over the transition reticulocyte-erythrocyte.In either cell the enzyme was not activated by catecholamines (epinephrine, norepinephrine, isoproterenol).     

Effects of pH and temperature on the interaction of an impermeant probe with surface proteins of the human red blood cell.

The conformation of the outer surface of the human red cell membrane has been studied under various conditions using the impermeant probe [125I]diazodiiodosulfanilic acid. At least seven polypeptides were labeled by the reagent, including the three extractable glycoproteins separable by the electrophoretic method employed. The Mr = 43,000 protein band was shown to contain two labeled species, one a glycoprotein, in addition to its major constituent, red cell actin. The extent and pattern of labeling were very sensitive to changes in pH and temperature. Total labeling increased with increasing pH and was greater at 4 degrees C than 37 degrees C. Binding of the probe to the Mr = 90,000 polypeptide and the major glycoprotein were relatively increased with increasing pH and temperature while opposite effects were observed for the Mr = 43,000 peptide(s). The pH effects on external membrane labeling were rapidly reversible. Results were similar in cells of different densities, suggesting that the pH and temperature effects were not related to cell age. The data presented emphasize the lability of membrane conformation and reactivity and thus the necessity to consider carefully the conditions of labeling in interpretation of studies using impermeant probes.     

Preparative purification of pig red blood cell hexokinase type III using a new efficient chromatographic support.

In this paper we report the purification of pig erythrocyte hexokinase type III, at preparative level, using 52 liters of starting material (hemolysate). This was possible using a new efficient anion exchanger support, the Toyopearl DEAE 650 M which allows completely to change the strategy of removing hemoglobin from hemolysates, permitting to handle large amounts of starting material and reducing work would have required months using conventional anion exchanger supports, to only 2-3 days. Furthermore, we have tested the binding of other red blood cell enzymes to the Toyopearl DEAE 650 M, showing a wider potential use of this chromatographic support for their purification at a preparative level.     

Effects of chitooligosaccharides on human red blood cell morphology and membrane protein structure

Recent studies of chitosan have increased the interest in its conversion to chitooligosaccharides (COSs) because these compounds are water-soluble and have potential use in several biomedical applications. Furthermore, such oligomers may be more advantageous than chitosans because of their much higher absorption profiles at the intestinal level, which permit their facilitated access to systemic circulation and potential distribution throughout the entire human body. In that perspective, it is important to clarify their effect on blood further, namely, on human red blood cells (RBCs). The aim of this work was thus to study the effect of two COS mixtures with different molecular weight (MW) ranges, <3 and <5 kDa, at various concentrations (5.0-0.005 mg/mL) on human RBCs. The interactions of these two mixtures with RBC membrane proteins and with hemoglobin were assessed, and the RBC morphology and surface structure were analyzed by optical microscopy (OM) and atomic force microscopy (AFM). In the presence of either COS mixture, no significant hemolysis was observed; however, at COS concentrations >0.1 mg/mL, changes in membrane binding hemoglobin were observed. Membrane protein changes were also observed with increasing COS concentration, including a reduction in both alpha- and beta-spectrin and in band 3 protein, and the development of three new protein bands: peroxiredoxin 2, calmodulin, and hemoglobin chains. Morphologic evaluation by OM showed that at high concentrations COSs interact with RBCs, leading to RBC adhesion, aggregation, or both. An increase in the roughness of the RBC surface with increasing COS concentration was observed by AFM. Overall, these findings suggest that COS damage to RBCs was dependent on the COS MW and concentration, and significant damage resulted from either a higher MW or a greater concentration (>0.1 mg/mL).